Preparation that contains oligosaccharides and probiotics

ABSTRACT

A preparation having a health-promoting action for the prevention and/or treatment of disorders of the digestive tract, contains one or more probiotics and one or more non-digestible oligosaccharides. The probiotics are preferably chosen from bacterial strains such as a strain of a  Lactobacillus  or a  Bifido bacterium  species and from yeast strains such as a strain of a  Saccharomyces  species.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a preparation which has ahealth-promoting action, in particular for the prevention and/ortreatment of disorders of the digestive tract, more particularly of theintestines.

The application relates in particular to a preparation of this typewhich contains probiotics and non-digestible oligosaccharides.

2. Description of the Related Art

It is known that certain microorganisms have both a prophylactic and atherapeutic effect on intestinal diseases, such as intestinalinfections. When these microorganisms are administered to humans oranimals they will compete with pathogenic bacteria for nutrients and/oradhesion sites on the intestinal wall, as a result of which the numberof pathogenic bacteria will decrease and infections are prevented orreduced. Such microorganisms are generally designated by the term“probiotics”.

If these microorganisms are to have an optimum action they must reachthe intestines alive. A further beneficial effect of the administrationof live microorganisms to the intestines is, for example, that they areable to ferment the oligosaccharides present in the intestines, whereby,for example, fatty acids with short chains are formed.

SUMMARY OF THE INVENTION

In addition it is advantageous on economic grounds if as manymicroorganisms as possible reach the intestines alive. With thecustomary preparations which contain probiotics the percentage ofmicroorganisms that reaches the intestines alive is frequently low.

It is therefore an object of the present invention to provide apreparation that contains such probiotics with which a high percentageof the microorganisms administered reaches the intestines alive.

It is also an object of the present invention to provide a preparationthat can be used for the treatment of disorders of the intestines and/orthat can be used for prophylactic treatment of the intestines.

The present invention now relates to a preparation having ahealth-promoting action, in particular for the prevention and/ortreatment of disorders of the digestive tract, more particularly of theintestines, which contains probiotics and non-digestibleoligosaccharides.

Without wishing to be tied to any theory, it is assumed that theoligosaccharides form a substrate for the probiotics, as a result ofwhich the likelihood that said microorganisms reach the intestines aliveincreases and as a result of which the likelihood that they are able, incombination with the oligosaccharides present in the intestines, toexert their beneficial action increases. The oligosaccharides couldtherefore also be designated as “prebiotics”.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The probiotics and non-digestible oligosaccharides are present in thepreparation in a ratio of 1 to 5 g oligosaccharides per 10⁸ to 10¹¹cells of the probiotic.

The oligosaccharides used are chosen from the so-called “non-digestibleoligosaccharides”, that is to say oligosaccharides which are notabsorbed by the human or animal body. These oligosaccharides as a rulehave a degree of polymerisation of 2 to 20. This implies that theoligosaccharides consist of 2 to 20 monosaccharide units. In the case ofa degree of polymerisation of less than 2, that is to say in the case ofthe monosaccharide, the preparation is not effective since suchmonosaccharides are absorbed by the human or animal body. At a degree ofpolymerisation of greater than 20 the oligosaccharides lose theirbeneficial action.

Preferably the oligosaccharides have a degree of polymerisation of 2 to10, more preferentially of 2 to 6, more particularly 3 to 10 and morepreferentially 3 to 6.

Where reference is made in the present application to oligosaccharidesthis term is used to refer both to oligosaccharides having one specificchain length and to mixtures of oligosaccharides having different chainlengths. However, a mixture of oligosaccharides having different chainlengths is preferred.

Furthermore, the oligosaccharides usually do not consist entirely ofdisaccharides. The disaccharide content is usually less than 90% andsometimes less than 60%.

The average degree of polymerisation is as a rule more than 2.1, usuallymore than 2.5.

The oligosaccharides used in the invention are, as a rule, furthermoreso chosen that they are at least 20% usable as substrate for theprobiotic microorganisms present in the preparation, as determined byhigh performance anion exchange chromatography.

Examples of suitable oligosaccharides are transgalacto-oligosaccharides(TOS), fructo-oligosaccharides (FOS) and combinations thereof.

It is particularly advantageous if the oligosaccharides are added to thepreparation in the form of a hydrolysis product of one or morepolysaccharides, for example chosen from β-(arabino)galactans,β-(arabino)xylans, β-glucans, β-glucomannans, β-galactomannans,α-arabans, inulin and combinations thereof. In addition to theoligosaccharides, such a hydrolysis product can also contains yetfurther components, such as monosaccharides and saccharides having adegree of polymerisation higher than 20. However, the hydrolysis productmust contain at least 50% non-digestible oligosaccharides, preferably atleast 70%.

The polysaccharides which are preferably hydrolysed areβ-(arabino)galactans, β-mannans, and xylans.

It is also possible to use a hydrolysis product of one or more fibreswhich are mainly made up of the abovementioned oligosaccharides, such asfibres from oats, wheat, potatoes, sugar beet, soya polysaccharides andthe like.

The hydrolysis of the polysaccharide(s) and/or the fibre(s) can becarried out in a manner known per se, for example by the use of suitableenzymes.

The concentration of oligosaccharides in the preparation is such that0.5 to 20 gram per day can be administered. If desired, thisadministration can be spread over the day, as long as the total quantityof the oligosaccharides administered remains in the range describedabove. In general the preparation will be administered 2 to 4 times perday.

In general the oligosaccharides will make up 5 wt. % to 50 wt. % of thetotal preparation.

The probiotics suitable for the present invention are generally known.They comprise, in general, one or more bacterial strains suitable foruse in food preparations, such as lactic acid bacteria suitable for usein food preparations, or one or more yeast strains suitable for use infood preparations, or a combination thereof. These probiotics willusually have GRAS (“Generally Recognised As Safe”) status.

Suitable bacterial strains are, for example, chosen from those which aredescribed in EP-A-0 904 784 in the name of the Applicant. Other possibleprobiotics are the Pediococci, Propionibacteria or Leuconostoc species.The Lactobacillus and Bifidobacterium genera and combinations thereofare to be preferred.

The Bifidobacterium strain used can be any strain which is suitable for,and preferably is also approved for, administration to humans andanimals, such as Bifidobacterium, bifidum, Bifidobacterium breve,Bifidobacterium lactis or Bifidobacterium longum, or a combinationthereof.

The Lactobacillus strain is preferably so chosen that this producesmainly, preferably exclusively, dextrorotatary (L+) lactate. What ismeant by this is that the lactate produced is less than 5%, preferablyless than 2%. laevorotatory lactate. It is, of course, possible that themicroorganism produces other metabolites in addition to lactate and thebeneficial action of the microorganism can (also) be based on theformation of these further metabolites.

Examples of these are Lactobacillus acidophilus, Lactobacillus brevis,Lactobacillus (para)casei, Lactobacillus fermentum, Lactobacillusplantarum and Lactobacillus rhamnosus, and combinations thereof.

The yeast strain used in the invention can be any strain which issuitable for, and preferably is also approved for, administration tohumans and animals, such as of the genus Saccharomyces. Examples ofsuitable Saccharomyces species are Saccharomyces cerevisiae andSaccharomyces boulardii.

The yeast in the preparation can be alive or dead. Both dead and liveyeast contains a high content of mannoproteins, which are able toprevent the adhesion of bacteria to the intestinal wall to a largeextent. Administration of dead yeast offers advantages in the case ofpeople who are suffering from inflammatory intestinal diseases.

If the yeast is used in dead form, at least one other live microorganismmust be used. Said live organism can once again be chosen from both theLactobacillus, Bifidobacterium, Pediococci, Propionibacteria andLeuconostoc strains, but can also be live Saccharomyces.

It is possible to use both a single microorganism and a mixture ofmicroorganisms.

The total concentration of the probiotics is 10⁶ to 10¹², preferably 10⁹to 10¹⁰, live cells per gram of total product. If a combination ofmicroorganisms is used, the minimum concentration of each of themicroorganisms must be such that there can still be said to be liveorganisms, that is to say at least approximately 10 per gram of product.The total concentration of microorganisms must always be within theabove specified range of 10⁶ to 10¹² live cells per gram of totalproduct.

If dead Saccharomyces cerevisae is also used, this is administered in aquantity of 0.5 to 5 g per day.

The combination of Lactobacillus rhamnosus withtransgalacto-oligosaccharide or hydrolysis products of (potato) galactanis found to be a particularly suitable combination of oligosaccharideand probiotic.

The suitability of a specific oligosaccharide for a specific probioticcan be determined by determining the capacity of the microorganismconcerned to ferment said oligosaccharide or said oligosaccharidefraction. A specific method for this is given below with the examples.

The administration forms of the preparations according to the inventionare as a rule analogous to those which have been described in EP-A-0 904784 in the name of the Applicant, the contents of which are incorporatedherein by reference. In this context it must be pointed out thataccording to the present invention also only one probiotic microorganism(including a yeast) can be present and that in addition to the one ormore probiotic microorganisms one or more oligosaccharides can also beincorporated, in the quantities specified above.

The preparations according to the present invention can, among others,be administered in the form of a nutritional supplement, total nutritionand clinical nutrition. Reference is likewise made to EP-A-0 904 784 forthe specific additives which are added to such foods and the preparationand applications of such foods.

The probiotics are preferably added to the preparation in (freeze-)driedform. It is also possible to produce liquid preparations, but these mustbe stored cool. Furthermore, one or more of the microorganisms can beused in encapsulated form, for example in order to improve the shelflife.

If the preparation according to the invention is used as a nutritionalsupplement, it can be administered as such, can be mixed with a suitabledrinkable liquid, such as water, yoghurt, milk or fruit juice, or can bemixed with solid or liquid food. In this context the nutritionalsupplement can be in the form of tablets, capsules, powders, sachets,pastilles, sweets, bars and corresponding administration forms, usuallyin the form of a unit dose.

A supplement according to the invention can, for example, have thefollowing composition:

probiotics: 10-40 wt. % oligosaccharides: 40-80 wt. % further additives: 0-40 wt. %,

The preparation can also be in the form of a food preparation that issuitable for direct consumption, such as total or clinical nutrition.This can be either oral nutrition or nutrition for administration via atube or catheter.

Such foods can be in solid form or liquid/drinkable form and can containall customary additives for (total and/or clinical) nutrition, includingproteins, vitamins, minerals, trace elements and the like.

A total nutrition according to the invention can, for example, have thefollowing composition:

probiotics: 0.1-10 wt. % oligosaccharides:   1-20 wt. % furtheradditives:  75-95 wt. %,

further additives: 75-95 wt. %

to a total of 100 wt. %.

According to a particular embodiment, the preparation according to theinvention is in the form of a (supplement for a) baby food or anutritional supplement for babies.

The preparations described above can be used for the same applicationsas those described in EP-A-0 904 784 in the name of the Applicant, inparticular in the treatment of disorders of the intestines, such asdiarrhea, such as can arise when travelling or after treatment withantibiotics, or which results from food poisoning. Another applicationis in the treatment of inflammatory bowel diseases (IBD), such ascolitis ulcerosa and Crohn's disease. The preparations according to theinvention are also suitable for patients who have a food allergy, suchas an allergy to cow's milk or to gluten.

The probiotics and non-digestible oligosaccharides can also be used inbaby food to prevent or treat intestinal problems.

The invention will now be explained with the aid of the followingnon-limiting examples.

EXAMPLES

To determine suitable combinations of oligosaccharides and probioticsthe microorganisms listed below were tested to determine their capacityto ferment structurally different oligosaccharide fractions. Strainspre-cultured in liquid medium based on thioglycolate (Oxoid, UnipathLtd, Basingstoke, Hampshire, UK) were subjected to sub-culture inthioglycolate to which 0.5% (m/V) oligosaccharides were added. Thesugar-free thioglycolate medium and the oligosaccharide solutions weresterilised separately for 15 minutes at 121° C.

Following anaerobic incubation for 48 hours at 37° C. the pH wasmeasured with the aid of a micro-pH meter (Sentron, Roden, TheNetherlands). The changes in the residual oligosaccharide content andthe formation of reaction products were determined using HPAEC (highperformance anionic exchange chromatography). For HPAEC analysis thecultures were centrifuged and the supematant liquor was diluted 10-foldwith H₂O and boiled for 5 minutes to stop the enzymatic activity. Thepurity of the stains was checked before and after fermentation.

The HPAEC system consisted of a Dionex Bio-LC GPM-II quaternary gradientmodule (Dionex Corporation. Sunnyval, Calif., USA) equipped with aDionex Carbopac PA-100 column (4*250 mm) in combination with a CarbopacPA-100 guard column (3*25 mm). The samples (20 μl) were injected using aSpectra Physics SP8880 autosampler. The oligomers were analysed using agradient of sodium acetate in 100 mmol.1⁻¹ NaOH.

The results of these experiments are given below in the table for anumber of combinations of probiotics and substrates, where:

Substrate Hydrolysis product of: Arabino- Bacteria galactans ArabansArabinoxylans Fructans Bi. Breve ++ + − ++ Bi. Longum ++ ++ + ++ Bi.adolescentis ++ + ++ ++ L. acidophilus ++ − − + L. fermentum ++ − − + K.pnuemoniae ++ − − + C. perfingens − − − ++

A few examples of preparations according to the present invention aregiven below.

Example I Supplement

A suspension of Lactobacillus rhamnosus ATCC 7469 (Lb) was freeze-dried,a powder being obtained which contained at least 10⁹ viable cells pergram powder. Transgalacto-oligosaccharides (TOS), obtained from lactose(Borculo Whey Products), were dissolved in water at 40° C. to a solidscontent of 25% and this solution was spray-dried. The two powders weremixed in a TOS/Lb ratio of 4/1 until a homogeneous product was obtained.Sachets were filled with 2-5 g of this mixture, depending on the dosageregime (5 g for one sachet per day; 3 g for two sachets per day). Thecontents of one sachet can, for example, be taken mixed in a glass oforange juice or milk.

Example II Synbiotic Bar

A 23 g bar was prepared from 4.0 g oat flakes, 4.0 g wheat flakes, 3.0 gpuffed rice, 1.0 g crushed hazelnuts, 0.25 g honey, 3.0 g raisins, 1.5 gmaltodextrin, 1.0 g freeze-dried Lb rhamnosus, 0.5 g baker's yeast(Saccharomyces cerevisiae; Gist Brocades) and 5.0 gtransgalacto-oligosaccharides.

Example III Method for Hydrolysing Vegetable Fibres

A 20% suspension of fibres, for example from wheat, potatoes, oats, soyapolysaccharides, carob gum or sugar beet, in water was prepared. Thesesources of fibre are commercially available. The suspension was heatedto a temperature of between 20 and 50° C. (preferably 35-45° C.), afterwhich enzymes were added in a quantity of one part enzyme per 5-500parts (m/m) substrate. The choice of the type of enzyme is dependent onthe type of polysaccharide. Examples of suitable enzymes are NovofermPectinex Ultra s.p.-L, Pentopan and Ultra.s.p. (NOVO).

After 0.5-4 hours the reaction was terminated by heating, after whichthe solution thus obtained can be used as the oligosaccharide fractionin the preparations according to the invention, optionally after furtherfiltration/purification or after concentrating.

Example IV Synbiotic Mixture for Mixing with a Complete Enteral ClinicalNutrition

A mother batch of a powder mixture was prepared in accordance with themethod of Example I. The powder consisted of 20% hydrolysed wheatarabinoxylans, 20% hydrolysed potato arabinogalactans, 20% hydrolysedcarob gum, 20% hydrolysed sugar beet fibre (arabans), 15% hydrolysed oatfibres (glucans) and 5% Bifidobacterium longum. 5 g of the powdermixture is placed in a sachet. The contents of this sachet can be addedto a standard enteral clinical nutrition a maximum of 30 min before use.

Example V Synbiotic Powder Mixture for Fortifying Baby Food

A synbiotic mixture was prepared in accordance with the method ofExample I. The composition contains 10% bakers yeast (Gist Brocades),40% mannoproteins, obtained from yeast, 25% inulin and 25% raffinose.

Example VI Sweet that Contains a Synbiotic Mixture

A 2 g sweet was prepared starting from 58% digestible carbohydrates(glucose Syrup), 35% TOS, 4% Lactobacillus rhamnosus ATCC 7469, and 2%flavourings and colourants.

What is claimed is:
 1. A nutritional supplement for the preventionand/or treatment of disorders of the digestive tract, which comprisesprobiotics comprising a bacterial strain selected from the groupconsisting of Lactobacillus, Bifidobacterium and Propionibacteriumstrains and at least one yeast strain and 40-80 weight % of one or moreoligosaccharides that are non-digestible by humans, said supplementbeing in dried form.
 2. The supplement according to claim 1, wherein theone or more oligosaccharides have a degree of polymerization of 2 to 20.3. The supplement according to claim 2, wherein the oligosaccharideshave a degree of polymerization of 2 to
 10. 4. The supplement accordingto claim 2, wherein the oligosaccharides have a degree of polymerizationof 2 to
 6. 5. The supplement according to claim 1, wherein the one ormore oligosaccharides have been obtained by the hydrolysis of one ormore polysaccharides selected from the group consisting ofβ-(arabino)galactans, β-(arabino)xylans, β-glucans, β-glucomannans,β-galactomannans, α-arabans, inulin and combinations thereof.
 6. Thesupplement according to claim 2, wherein the oligosaccharides have beenobtained by the hydrolysis of one or more polysaccharides selected fromthe group consisting of β-(arabino)galactans, β-(arabino)xylans,β-glucans, β-glucomannans, β-galactomannans, α-arabans, inulin andcombinations thereof.
 7. The supplement according to claim 5, whereinthe one or more polysaccharides are selected from the group consistingof β-(arabino)galactans, β-glucomannans, β-(arabino)xylans andcombinations thereof.
 8. The supplement according to claim 1, whereinthe oligosaccharides are produced by hydrolysis of one or more fibersoriginating from oats, wheat, potatoes, sugar beet, and soyapolysaccharides.
 9. The supplement according to claim 2, wherein theoligosaccharides are produced by hydrolysis of one or more fibersoriginating from oats, wheat, potatoes, sugar beet, and soyapolysaccharide.
 10. The supplement according to claim 1, furthercomprising dead yeast cells.
 11. The supplement according to claim 1,wherein the ratio between the probiotics and the one or morenon-digestible oligosaccharides is in the range of 1 to 5 goligosaccharides per 10⁸ to 10¹¹ cells of the probiotics.
 12. Thesupplement according to claim 1, which contains the probiotics in aconcentration of 10⁷ to 10¹¹ live cells per gram of total product.
 13. Anutritional supplement for the prevention and/or treatment of disordersof the digestive tract, which comprises probiotics comprising a lacticacid bacterial strain and a yeast strain of a Saccharomyces species and40-80 weight % of one or more oligo-saccharides that are non-digestibleby humans, said supplement being in dried form.